Potomac horse fever isolates

ABSTRACT

The present invention discloses novel isolates of  Neorickettsia risticii , compositions comprising such isolates, vaccines and methods for using such vaccines against Potomac Horse Fever.

RELATED APPLICATIONS

This application depends for priority on U.S. Provisional ApplicationNo. 61/378,261 filed Aug. 30, 2010 and U.S. Provisional Application No.61/381,326 filed Sep. 9, 2010, both of which are hereby incorporated byreference in their entireties.

FIELD OF INVENTION

The present invention relates to novel isolates of Neorickettsiaristicii and compositions comprising such isolates, and methods of usingsuch compositions in vaccines against Potomac Horse Fever.

BACKGROUND

Potomac Horse Fever (PHF), an acute infectious disease of horses, wasreportedly first recognized in 1979 in the region of the Potomac Riverin Maryland and Virginia. The disease is also identified as EquineMonocytic Ehrlichiosis and Equine Intestinal Ehrlichiosis. The causativeagent is a gram-negative, obligate intracellular bacterium which wasfirst identified in 1984 as Ehrlichia risticii (E. risticii), but hasbeen renamed Neorickettsia risticii (N. risticii). The disease ischaracterized by a wide variety of intestinal symptoms, along withelevated temperature and inflamed mucus membranes. In many cases itresults in severe pain, and sometimes death.

The bacteria infect the enterocytes of the small and large intestine,resulting in acute colitis syndrome, and producing symptoms of mildcolic, fever, depression, anorexia and diarrhea in horses of all ages.The disease can also cause abortion in pregnant mares, laminitis, anddeath. N. risticii has been isolated from trematodes infecting freshwater snails, and from caddisflies, mayflies, damselflies, dragonfliesand stoneflies. The route of infection appears to be inadvertentingestion of the aquatic insects carrying N. risticii, and theincubation period is 10 to 18 days.

An antigen for use in an assay to detect the presence of N. risticii hasbeen claimed in U.S. Pat. No. 4,759,927, which is herein incorporated byreference in its entirety. Its source was later identified as the 25-Dstrain. A second strain identified as 90-12, was disclosed in U.S. Pat.No. 6,375,954, which is herein incorporated by reference in itsentirety. U.S. Pat. No. 6,375,954 claims a method for protecting againstN. risticii by administering a particular 90-12 protein antigen. Equinevaccines are commercially available, but provide only partial or noprotection against newer strains of N. risticii, and against strains wehave isolated relating to the present invention.

The citation of any reference herein should not be construed as anadmission that such reference is available as “prior art” to the instantapplication.

SUMMARY OF THE INVENTION

The present invention provides three novel strains of N. risticii thatinfect horses, and possibly other mammals. We have identified thesethree novel N. risticii isolates as N. risticii Oregon (OR), N. risticiiNew York (NY) and N. risticii Michigan (MI), and deposited them with theATCC, Manassas, Va., USA, as N. risticii Oregon (ATCC No. PTA-11232), N.risticii New York (ATCC No. PTA-11231) and N. risticii Michigan (ATCCNo. PTA-11404). We have characterized these strains and have shown themto be biologically and structurally distinguishable from previouslyknown strains.

The invention also provides novel protein antigens that characterize thenew strains, as well as nucleic acids encoding these protein antigens,expression vectors that comprise such nucleic acids and express theprotein antigens, vaccines comprising the new strains and/or the proteinantigens, and/or the expression vectors, compounds comprising theirprotective antigens, methods for protecting animals, methods forproducing the new strains and assays for detecting the novel strains.There is therefore a need for new vaccines against Potomac Horse fever.

These and other aspects of the present invention will be betterappreciated by reference to the following figures and DetailedDescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a Western Blot showing antibody reactivity and antigenbanding with rabbit serum against the recombinant 85 kD strain specificantigen (SSA) of the N. ristcii 90-12 strain, FEF cells used topropagate N. risticii, and the 90-12, OR, NY, MI and 25-D strains.

FIG. 2 depicts a Western Blot showing antibody reactivity and antigenbanding with horse serum against the recombinant 85 kD SSA of the N.ristcii 90-12 strain, FEF cells used to propagate N. risticii, and the90-12, OR, NY, MI and 25-D strains.

FIG. 3 depicts a Western Blot showing reactivity, or lack thereof, ofthe same antigens against seronegative horse serum.

FIG. 4 depicts a strain specific antigen (SSA) amino acid sequencealignment for the SSA 1 proteins of six N. risticii isolates. From thetop to bottom:

-   -   N. risticci ILL stain-CP001431, SEQ ID NO: 1    -   N. risticci Oregon, SEQ ID NO: 2    -   N. risticci 25-D, SEQ ID NO: 3    -   N. risticci, 90-12 SEQ ID NO: 4    -   N. risticci Michigan, SEQ ID NO: 5    -   N. risticci New York, SEQ ID NO: 6

FIG. 5 depicts a SSA amino acid sequence alignment for the SSA 2 proteinof six N. risticii isolates. From the top to bottom:

-   -   N. risticci ILL stain-CP001431, SEQ ID NO: 7    -   N. risticci New York, SEQ ID NO: 8    -   N. risticci 90-12, SEQ ID NO: 9    -   N. risticci, 25-D, SEQ ID NO: 10    -   N. risticci Oregon, SEQ ID NO: 11    -   N. risticci, Michigan, SEQ ID NO: 12

FIG. 6 depicts a SSA amino acid sequence alignment for SSA 3 protein ofsix N. risticii isolates. From the top to bottom:

-   -   N. risticci ILL stain-CP001431, SEQ ID NO: 13    -   N. risticci New York, SEQ ID NO: 14    -   N. risticci 90-12, SEQ ID NO: 15    -   N. risticci, 25-D, SEQ ID NO: 16    -   N. risticci, Michigan, SEQ ID NO: 17    -   N. risticci, Oregon, SEQ ID NO: 18

FIG. 7 depicts a SSA nucleic acid sequence alignment for the genesencoding SSA 3 of six N. risticii isolates. From the top to bottom:

-   -   N. risticci ILL stain-CP001431, SEQ ID NO: 19    -   N. risticci New York, SEQ ID NO: 20    -   N. risticci 90-12, SEQ ID NO: 21    -   N. risticci, 25-D, SEQ ID NO: 22    -   N. risticci, Michigan, SEQ ID NO: 23    -   N. risticci, Oregon, SEQ ID NO: 24

FIG. 8 depicts the nucleic acid sequence (SEQ ID NO: 25) for the codingsequence of the SSA#1 protein of N. risticii Oregon.

FIG. 9 depicts the nucleic acid sequence (SEQ ID NO: 26) for the codingsequence of the SSA#2 protein of N. risticii Oregon.

FIG. 10 depicts the nucleic acid sequence (SEQ ID NO: 27) for the codingsequence of the SSA#1 protein of N. risticii New York.

FIG. 11 depicts the nucleic acid sequence (SEQ ID NO: 28) for the codingsequence of the SSA#2 protein of N. risitcii New York.

FIG. 12 depicts the nucleic acid sequence (SEQ ID NO: 29) for the codingsequence of the SSA#1 protein of N. risticii Michigan.

FIG. 13 depicts the nucleic acid sequence (SEQ ID NO: 30) for the codingsequence of the SSA#2 protein of N. risticii Michigan.

FIG. 14 depicts the nucleic acid sequence (SEQ ID NO: 31) for the codingsequence of the SSA#1 protein of N. risticii Illinois (CP001431).

FIG. 15 depicts the nucleic acid sequence (SEQ ID NO: 32) for the codingsequence of the SSA#2 protein of N. risticii Illinois (CP001431).

DETAILED DESCRIPTION OF THE INVENTION

Commercially available vaccines presently comprise antigens from theIllinois strain of N. risticii. Commercially available vaccines includePOTOMAVAC™ from Merial, Athens, Ga. and POTOMACGUARD™ from Pfizer Inc,Fort Dodge, Iowa. The available vaccines do not presently provide fullprotection against all current Potomac Horse Fever (PHF) outbreaks.Therefore, we have sought to identify currently circulating strains thatare immunogenically distinguishable from the known vaccine strains. Forthis purpose, we have solicited serum samples from animals showingclinical signs of PHF, from which we have isolated and characterized thethree new N. risticii strains of the invention. These three new strains,N. risticii Oregon, N. risticii New York and N. risticii Michigan, canbe distinguished both by their strain specific antigens (SSA's), i.e.,expressed surface antigens that characterize the different strains of N.risticii, and by the failure of available vaccines to fully protectanimals against infection upon challenge with these novel strains.

The novel Oregon strain was isolated from a blood sample from a horse inOregon that had been immunized with a commercially available vaccine,but still exhibited clinical signs of PHF. We found that horsesimmunized with the N. risticii 90-12 strain were not protected whenchallenged with the newly isolated N. risticii Oregon strain. Inaddition, N. risticii Oregon did not to react with monoclonal antibodiesraised against the 90-12 strain.

The novel New York (NY) strain was isolated from the blood sample of ahorse from New York State exhibiting signs of PHF. We found that horsesimmunized with the N. risticii 90-12 strain showed a significantreduction in clinical disease and bacteremia upon heterologous challengewith the N. risticii New York strain. N. risticii New York does reactwith a monoclonal antibody to the 90-12.

The novel Michigan (MI) strain was isolated from the blood sample of ahorse from Michigan exhibiting signs of PHF. The MI strain is shown tobe unique by its antigen banding pattern (FIGS. 1 and 2) and by its SSA#1 protein sequence when compared with the other isolates.

N. risticii bacteria are typically characterized by their strainspecific antigens, and the Oregon strain has a SSA #3 of approximately50 kDa, the New York strain has a SSA #3 of approximately 55 kDa, andthe Michigan strain has a SSA#3 of approximately 60 kDa, whereas the90-12 strain has a SSA #3 of approximately 85 kDa.

In addition to the novel strains of N. risticii and their antigens, thepresent invention also relates to immunogenic compositions and vaccines.The therapeutic agent (also referred to as the antigen, active agent, orthe immunogenic composition) that can serve as the basis for a vaccinecan be one or more of the following:

a) harvested cultures of host cells that are infected with N. risticiibacteria;

b) extracts or fractions of (a) that are enhanced with respect to theconcentration of the N. risticii bacteria contained within the infectedhost cells;

c) N. risticii bacteria enhanced extracts of (a) that contain remnantsof the host cells;

d) isolated and purified N. risticii bacterial extracts of (a) that donot contain remnants of the host cells;

e) attenuated or inactivated bacteria;

f) isolated bacterial immunogens;

g) recombinant N. risticii proteins;

h) recombinant expression vectors that comprise nucleotide sequences,under the control of one or more promoters, that encode one or morerecombinant N. risticii proteins (e.g., N. risticii strain specificantigens) which can be expressed by the recombinant expression vector;and

i) N. risticii strain specific antigens.

In some embodiments an N. risticii isolate of the present invention,encodes an SSA #1 protein comprising an amino acid sequence thatcomprises 80% or greater, 90% or greater, 95% or greater, 98% orgreater, and/or 99% or greater identity with the amino acid sequence ofSEQ ID NO: 2. In some embodiments an N. risticii isolate of the presentinvention, encodes an SSA #2 protein comprising an amino acid sequencethat comprises 80% or greater, 90% or greater, 95% or greater, 98% orgreater, and/or 99% or greater identity with the amino acid sequence ofSEQ ID NO: 11. In some embodiments an N. risticii isolate of the presentinvention, encodes an SSA #3 protein comprising an amino acid sequencethat comprises 90% or greater, 95% or greater, 98% or greater, and/or99% or greater identity with the amino acid sequence of SEQ ID NO: 18.In other embodiments an N. risticii isolate of the present invention,encodes an SSA #1 protein comprising an amino acid sequence thatcomprises 80% or greater, 90% or greater, 95% or greater, 98% orgreater, and/or 99% or greater identity with the amino acid sequence ofSEQ ID NO: 2, encodes an SSA #2 protein comprising an amino acidsequence that comprises 80% or greater, 90% or greater, 95% or greater,98% or greater, and/or 99% or greater identity with the amino acidsequence of SEQ ID NO: 11, and encodes an SSA #3 protein comprising anamino acid sequence that comprises 90% or greater, 95% or greater, 98%or greater, and/or 99% or greater identity with the amino acid sequenceof SEQ ID NO: 18.

As used herein the following terms shall have the definitions set outbelow:

“Isolated” when used herein means removed from its naturally occurringenvironment. Hence, isolated N. risticii bacterial cells broadly includethose that have been removed from their naturally occurringenvironments, including without limitation arthropods, insects, infectedanimals and specimens from infected animals. Isolated N. risticiibacterial cells also include those that are contained within host cellsas described herein, or separated therefrom, as well as those that aresubstantially free of other microorganisms, e.g., in a culture.

“Isolated bacterial immunogens” refers to bacterial immunogens that havebeen completely or partially separated from their respective sourcebacteria. Compositions of isolated bacterial immunogens can include somewhole intact bacteria, portions or components of bacteria, whole intacthost cell, portions or components of host cells comprising bacterialantigens, as well as antigens produced by physical, chemical, biologicalor molecular biological processes.

“N. risticii bacterial immunogens” as used herein include wholebacteria, as well as parts thereof, including proteins (lipoproteins,membranous proteins, cytosolic proteins), immunogenic fragments of suchproteins, nucleic acids, lipids, saccharides, lipopolysaccharides orother biological molecules derived from the N. risticii bacteria. Theymay be present in live host cells and host cells that are killed orinactivated. The skilled artisan is generally familiar with techniquesby which bacteria or host cells can be killed or inactivated. Suchtechniques include physical, chemical and biological means. Non-limitingexamples of inactivation techniques include sonication, freeze-thawtechniques, pressure, treatment with heat, chemicals or enzymes.Non-limiting examples of chemical inactivation agents include treatmentwith binary ethyleneamine (BEA) and formalin (formaldehyde solution).Immunogens may also be the products of chemical, biological or molecularbiological processes.

As used herein one amino acid sequence is 100% “identical” to a secondamino acid sequence when the amino acid residues of both sequences areidentical. Accordingly, an amino acid sequence is 50% “identical” to asecond amino acid sequence when 50% of the amino acid residues of thetwo amino acid sequences are identical. The sequence comparison isperformed over a contiguous block of amino acid residues comprised by agiven protein, e.g., a protein, or a portion of the polypeptide beingcompared. In a particular embodiment, selected deletions or insertionsthat could otherwise alter the correspondence between the two amino acidsequences are taken into account.

As used herein, nucleotide and amino acid sequence percent identity canbe determined using C, MacVector (MacVector, Inc. Cary, N.C. 27519),Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) andthe Clustal W algorithm with the alignment default parameters, anddefault parameters for identity. These commercially available programscan also be used to determine sequence similarity using the same oranalogous default parameters. Alternatively, an Advanced Blast searchunder the default filter conditions can be used, e.g., using the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program using the default parameters.

As used herein, the term “vaccine(s)” means and refers to a product, theadministration of which is intended to elicit an immune response thatcan prevent and/or lessen the severity of one or more infectiousdiseases.

As used herein, an “immune response” refers to the subject animal'sactive immunity due to having received one or more vaccines. The immuneresponse can include the production of antibodies to the antigen orimmunogen present in the vaccine. “Immune response” in a subject refersto the development of a humoral immune response, a cellular immuneresponse, or a humoral and a cellular immune response to an antigen.Immune responses may be measured using standard immunoassays andneutralization assays, which are known in the art.

“Preventing infection” and like terms means to prevent or inhibit thereplication of the bacteria that cause the identified disease, toinhibit transmission of the bacteria or virus, to prevent the bacteriafrom establishing itself in its host animal or to alleviate the symptomsof the disease caused by infection. The treatment is consideredtherapeutic if there is a reduction in bacterial load.

“Protection,” “Protecting” and the like, as used herein with respect tobacteria, mean that the vaccine prevents or reduces the symptoms of thedisease caused by the organism from which the antigen(s) used in thevaccine is derived. The terms “protection,” “protecting” and the likealso mean that the vaccine may be used to “treat” the disease or one ofmore symptoms of the disease that already exists in a subject.

“Treating” refers to reversing, alleviating, inhibiting the progress of,or preventing a disorder, condition or disease to which such termapplies, or to preventing one or more symptoms of such disorder,condition or disease.

“Pharmaceutically acceptable” as used herein refers to substances (e.g.,adjuvants, immunostimulants, carriers, diluents, emulsifying orstabilizing agents) that are within the scope of sound medical judgment,suitable for use in contact with the tissues of subjects without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit-to-risk ratio, and effective for their intendeduse. Pharmaceutically acceptable substances do not interfere with theeffectiveness of the therapeutic agent.

A vaccine contains an antigen (or, “active agent,” “immunogen,”“therapeutic agent,” or “immunogenic composition”), including a hostcell infected with N. risticii bacteria, whole intact bacteria, orbacterial fractions or parts or biomolecules that act to stimulate theimmune system in an animal, particularly the SSA's of the novel strain.An antigen may be a live attenuated or killed preparation ofbacteria-infected host cells, live attenuated or killed bacteria, livingirradiated cells, crude fractions or purified bacterial immunogens. Avaccine can comprise enriched, isolated or purified antigen. Thevaccines can be made from inactivated or killed cultures of infectedhost cells, or inactivated or killed bacteria or alternatively, comprisea recombinant expression vector that comprises one or more nucleotidesequence, under the control of one or more promoters, that encode one ormore recombinant N. risticii proteins (e.g., N. risticii strain specificantigens) which can be expressed by the recombinant expression vector.

A vaccine of the present invention may also comprise a combination ofantigens from more than one N. risticii bacterial species and/or acombination of N. risticii antigens. For example, a vaccine of thepresent invention can comprise a combination of two or more of thefollowing strains: N, risticci Oregon, N. risticci 90-12, N. risticciIllinois, N. risticci New York, N. risticci 90-12, N. risticci, 25-D,and/or N. risticci, Michigan. In a particular embodiment of this type,the vaccine can comprise N. risticci Oregon, and N. risticci 90-12. Inanother such embodiment, the combination vaccine can comprise N.risticci Oregon, and N. risticci New York. In yet another embodiment,the combination vaccine can comprise N. risticci New York and N.risticci, Michigan. In still another embodiment, the vaccine cancomprise N. risticci Oregon, N. risticci 90-12, and N. risticci NewYork. All other such combinations are further envisioned by the presentinvention. In addition, any N. risticii vaccine of the present inventioncan further include antigens from other pathogens (e.g. viral, bacterialparasitical or fungal), as described further below.

Vaccines made from material cultured according to the present inventioncomprise a therapeutically effective amount of the antigen. In thecontext of this disclosure, a “therapeutically effective amount” refersto an amount of an antigen or vaccine that would induce an immuneresponse in an animal receiving the antigen or vaccine that is adequateto prevent or ameliorate signs or symptoms of disease caused byinfection with a N. risticii bacterium. Humoral immunity orcell-mediated immunity, or both humoral and cell-mediated immunity, maybe induced. The immunogenic response of an animal to a vaccine may beevaluated, e.g., indirectly through measurement of antibody titers, viamicroscopic analysis, or directly through monitoring signs and symptomsafter challenge with wild type strain. The protective immunity conferredby a vaccine may be evaluated by measuring, e.g., reduction in clinicalsigns such as mortality, morbidity, body temperature and overallphysical condition and overall health and performance of the subject.The amount of a vaccine that is therapeutically effective may varydepending on the particular antigen used, or the condition of thesubject, and may be determined by one skilled in the art through wellknown means.

The novel N. risticii strains according to the present invention canalso be used to diagnose diseases or illnesses caused by N. risticiibacteria. Non-limiting examples of such diagnostic applications includeuse of bacterial fractions, proteins or other biomolecules in antibodybinding assays. The bacterial fractions, proteins or other biomoleculesmay also be used to generate polyclonal or monoclonal antibodies forsuch assays.

Host Cell Growth

Host cells for culturing bacterial organisms according to the presentinvention are first prepared prior to infecting with the desiredbacterial organism. Examples of appropriate cell lines include: felineembryonic fibroblast cells, mouse macrophage P388D1 cells (ATCC No.: TIB63), or a human histiocyte (HH) cell line (ATCC No. U937).

In one embodiment a sample of an isolated feline embryonic fibroblastcell line is seeded into media for either suspended or adherent growth.As used herein, adherent growth conditions exist when a layer of cellscoats surfaces contained within the vessel in which the cells arecultured. The surfaces can include the interior surface of the vesselitself, or surfaces of glass or polymeric beads contained within thevessel to increase surface area. Microcarriers can also be used toincrease surface area and host cell growth. In contrast to adherentgrowth, host cells may also be grown in suspension, in which the hostcells need not bind to surfaces within the culturing vessel.

The skilled artisan is generally familiar with the varieties ofculturing media that may be used to grow the host cells. The host cellgrowth media may be derived from animals. Alternatively, the host cellgrowth media may be vegetable or yeast based, and may be animalprotein-free. The growth media may be derived from soy bean extracts orfrom other protein-rich plants or protein-rich plant food productsincluding, for example, legumes. Non-limiting examples of specific mediauseful for growing host cells include Dulbecco's Modified Eagle's Medium(D-MEM), Eagle's Minimal Essential Media (MEM), Glasgow-MinimalEssential Media, RPMI1640, OptiMEM, and AIM V. The growth media maycontain or be supplemented with fetal bovine serum (FBS), tryptosesolution, lactos-albumin hydrosolate solution, L-glutamine, sodiumbicarbonate; lactalbumin hydrolysate, Polymyxin B, sodium pyruvate,glucose, and/or magnesium sulfate.

Fresh growth media may be fed or replenished to the host cells prior toor after infection or exposure of the host cells to the bacteria. Cellsmay be grown at 36-38° C. for 2-9 days at 5% CO₂.

Infecting the Host Cells

The host cells may be exposed to or infected with bacterial organisms bybringing the host cells into contact with other eukaryotic cells knownto be infected with the bacterial organisms. The skilled artisan isfamiliar with determining whether such other eukaryotic cells from amammal, for example, are infected with such bacterial organisms. Theinfected mammalian cells may be derived from any tissue, including thespleen, liver, pancreas, lungs, heart or other muscle tissue, brain,gall bladder, blood, kidneys, lymph nodes or stomach. The infectedmammalian cells may be prepared from a tissue extract via blenderhomogenization in an appropriate isotonic solution. The homogenate canthen be used to innoculate (i.e., infect) a culture of host cells,applied as a layer over the host cells or simply brought into contactwith them.

Alternatively, the host cells may be exposed to or infected withisolated bacterial organisms. The skilled artisan is familiar withtechniques of isolating such bacterial organisms, or can obtain stocksof isolated bacterial organisms from a biological depository.

The growth medium used to prepare host cells prior to contact withbacteria may be the same as the medium used to propagate the host cellsafter such contact. The bacteria-exposed (or infected) host cells may becultured for up to 95 days, up to 35 days, or for about 5 to 14 days, toachieve a titer of ≧1×10⁴ TCID₅₀ (Tissue Culture Infectious Dose), andthen the culture may be harvested and processed.

Harvesting

The bacteria infected host cells may be harvested by collecting thetissue cell culture fluids and/or cells. The host cells may be harvestedfrom the media (and the culture vessels) with the bacterial cellscontained with the walls of the host cells. Alternatively, duringharvesting the concentration of the bacteria may be enriched bytechniques that improve the liberation of the infective bacterial cellsfrom the growth substrate, e.g., sonication, freeze thawing, heating,pressure or chemical or selective enzymatic lysis of the eukaryotic hostcells. An enriched harvest of bacteria can include material that is freeof host cells or host cell material. Alternatively, an enriched harvestof bacteria can include material that contains host cells or host cellmaterial.

Inactivating

The skilled artisan is generally familiar with the techniques by whichbacteria or host cells can be killed or inactivated. Such techniquesinclude, physical, chemical and biological means. Non-limiting examplesof inactivation techniques include sonication, freeze-thaw techniques,pressure, treatment with heat, chemicals or enzymes. Non-limitingexamples of chemical inactivation agents include treatment with binaryethyleneimine (BEI), formalin (formaldehyde solution),beta-propiolactone, merthiolate, gluteraldehyde, sodium dodecyl sulfate,or the like, or a mixture thereof. The host cells can also beinactivated by heat or psoralen in the presence of ultraviolet light.These chemical inactivation agents or physical inactivation means canalso be used to inactivate the bacterial cells after their having beenextracted or separated from the host cells.

Formulating

The inactivated, infected host cells or enriched bacterial cells canserve as the antigen and may be formulated as a liquid suspension or maybe lyophilized for its use in the preparation of a vaccine againstdiseases caused by the organisms. Material cultured according to thepresent invention can be formulated with any pharmaceutically acceptableadjuvants, immunostimulants, carriers, diluents, emulsifying orstabilizing agents, non-limiting examples of which are discussed below.The skilled artisan, however, would recognize that other adjuvants,immunostimulants, carriers, diluents, emulsifying agents or stabilizingagents may be used in formulating vaccines based upon material culturedaccording to the present invention.

Adjuvants & Immunostimulants

An adjuvant in general is a substance that boosts the immune response ofthe target in a non-specific manner. Many different adjuvants are knownin the art. Non-limiting examples of adjuvants that may be used in theformulation of a vaccine made with material according to the presentinvention include aluminum salts (e.g., alum, aluminum hydroxide,aluminum phosphate, aluminum oxide), cholesterol, monophosphoryl lipid Aadjuvants, amphigen, tocophenols, monophosphenyl lipid A, muramyldipeptide, oil emulsions, glucans, carbomers, block copolymers, Avridinelipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinantor otherwise), cholera toxin, muramyl dipeptide, Freund's Completeand—Incomplete adjuvant, vitamin E, non-ionic block polymers andpolyamines such as dextransulphate, carbopol, pyran, saponins andsaponin derivatives, block co-polymers, and adjuvants such as thoseidentified in U.S. Pat. Nos. 4,578,269, 4,744,983, 5,254,339, which areall herein fully incorporated by reference. Non-limiting examples ofpeptides that can serve as adjuvants include muramyldipeptides,dimethylglycine, or tuftsin. Non-limiting examples of oils that canserve as adjuvants include mineral oils, vegetable oils, animal oils andemulsions thereof.

Vaccines made from material according to the present invention may beformulated as oil-in water emulsions, as water-in-oil emulsions or aswater-in-oil-in-water emulsions. Non-limiting examples of oil-in-wateremulsions include paraffin oil-in-water emulsions, or emulsions madefrom one or more of squalene, block copolymers of ethylene oxide andpropylene oxide, polysorbate surfactants, and/or threonyl analogs ofmuramyl dipeptide.

Oils used as adjuvants may be metabolizable by the subject receiving thevaccine such as vegetable or animal oils. Such oils typically consistlargely of mixtures of triacylglycerols, also known as triglycerides orneutral fats. These nonpolar, water insoluble substances are fatty acidtriesters of glycerol. Triacylglycerols differ according to the identityand placement of their three fatty acid residues.

Adjuvants may also consist of components that cannot be metabolized bythe body of the animal subject to which the emulsion is administered.Non-metabolizable oils suitable for use in the emulsions of the presentinvention include alkanes, alkenes, alkynes, and their correspondingacids and alcohols, the ethers and esters thereof, and mixtures thereof.The individual compounds of the oil may be light hydrocarbon compounds,e.g., compounds having 6 to 30 carbon atoms. The oil may besynthetically prepared or purified from petroleum products. Non-limitingexamples of non-metabolizable oils for use in the preparation ofvaccines based upon material cultured according to the present inventioninclude mineral oil, paraffin oil, and cycloparaffins, for example. Theterm “mineral oil” refers to a non-metabolizable adjuvant oil that is amixture of liquid hydrocarbons obtained from petrolatum via adistillation technique. The term is synonymous with “liquefiedparaffin,” “liquid petrolatum” and “white mineral oil.” The term is alsointended to include “light mineral oil,” i.e., oil which is similarlyobtained by distillation of petrolatum, but which has a slightly lowerspecific gravity than white mineral oil.

Other compounds capable of enhancing a humoral immunity response thatmay be used in the formulation of vaccines based upon material culturedaccording to the present invention include, without limitation, ethylenemaleic anhydrate (EMA) copolymer, latex emulsions of a copolymer ofstyrene with a mixture of acrylic acid and methacrylic acid.

In addition to the adjuvant, a vaccine based upon material culturedaccording to the present invention can include immunomodulatory agentssuch as, e.g., interleukins, interferons, or other cytokines (e.g.,Th1-related cytokines, such as interleukin-12 (IL-12), interleukin-18(IL-18), or gamma interferon).

The amount of adjuvant or immunostimulant added in a vaccine formulationbased upon material cultured according to the present invention dependson the nature of the adjuvant or immunostimulant itself. The skilledartisan is capable of selecting an amount that is sufficient to enhancean immune response to the bacterial immunizing agent.Carriers

Pharmaceutically acceptable carriers suitable for use in vaccinescomprising material according to the present invention may be anyconventional liquid carrier suitable for veterinary pharmaceuticalcompositions, including balanced salt solutions suitable for use intissue culture media. Pharmaceutically acceptable carriers areunderstood to be compounds that do not adversely effect the health ofthe animal to be vaccinated, at least not to the extent that the adverseeffect is worse than the effects seen when the animal is not vaccinated.Suitable carriers also include sterile water, saline, aqueous bufferssuch as PBS, solvents, diluents, isotonic agents, buffering agents,dextrose, ethanol, mannitol, sorbitol, lactose and glycerol, and thelike.

Vehicle

Vaccines formulated from material according to the present invention mayalso comprise a vehicle. A vehicle is a compound to which the hostcells, bacterial cells, or proteins, protein fragments, nucleic acids orparts thereof adhere, without being covalently bound to it. Non-limitingexamples of such vehicles include bio-microcapsules, micro-alginates,liposomes and macrosols. Some materials that serve as adjuvants can alsoserve as vehicles such as aluminum-hydroxide, aluminum phosphate,aluminum sulphate or aluminum oxide, silica, kaolin, and bentonite, allknown in the art.

Stabilizers

Often, a vaccine is mixed with stabilizers, e.g., to protectdegradation-prone components from being degraded, to enhance theshelf-life of the vaccine, or to improve freeze-drying efficiency.Non-limiting examples of stabilizers that may be added to vaccineformulations based upon material cultured according to the presentinvention include SPGA, skimmed milk, gelatins, bovine serum albumin,carbohydrates (e.g., sorbitol, mannitol, trehalose, starch, sucrose,dextran or glucose), proteins (e.g., albumin, casein or degradationproducts thereof), non-animal origin stabilizers, and buffers (e.g.,alkali metal phosphates).

Multivalent Vaccines

An immunogen according to the present invention may be formulated in avaccine comprising one or more additional antigens. Additionalimmunoactive component(s) may be whole parasites, bacteria or viruses(inactivated or modified live), or a fractionated portion or extractthereof (e.g., proteins, lipids, lipopolysacharides, carbohydrates ornucleic acids).

Where the immunogen according to the present invention is used in anequine vaccine, antigens from other pathogens may be added into theformulation. Non-limiting examples of other pathogens for whichadditional antigens may be added include one or more (including all) ofthe following: Tetanus, Rabies, Eastern Encephalomyelitis, WesternEncephalomyelitis, influenza virus, herpesvirus, West Nile virus(including a yellow fever virus/west nile virus chimeric flavivirus,live or killed, see e.g., US 2009/0246233, hereby incorporated byreference in its entirety) and Venezuelan Encephalomyelitis.Alternatively, a vaccine based upon material according to the presentinvention may be administered simultaneously with other live orinactivated vaccines.

Freeze-Drying/Reconstitution

For reasons of stability or economy, vaccines based upon materialcultured according to the present invention may be freeze-dried. Ingeneral this will enable prolonged storage at temperatures above 0° C.,e.g., at 4° C. Procedures for freeze-drying are known to persons skilledin the art. Equipment for freeze-drying at different scales is availablecommercially. To reconstitute the freeze-dried vaccine, it may besuspended in a physiologically acceptable diluent. Such diluents may beas simple as sterile water, a physiological salt solution or othercarrier as discussed above.

Dosaging

Vaccines based upon material according to the present invention may beformulated in a dosage unit form to facilitate administration and ensureuniformity of dosage. A dosage unit as it pertains to the vaccinecomposition refers to physically discrete units suitable as unitarydosages for animals, each unit containing a predetermined quantity ofbacterial immunogen calculated to produce the desired immunogenic effectin association with the required adjuvant system and carrier or vehicle.

The effective immunizing amount of bacterial immunogen can varydepending upon the chosen strain or strains and may be any amountsufficient to evoke a protective immune response. For example, amountswherein the dosage unit comprises at least about 1×10⁴ TCID₅₀inactivated bacterin are suitable.

Administering

Administration of the vaccine to a subject results in stimulating animmune response in the subject mammal. The route of administration forvaccines according to the present invention may be administered to themammalian target according to methods known in the art. Such methodsinclude, but are not limited to, intradermal, intramuscular,intraocular, intraperitoneal, intravenous, mucosal, oral, oronasal, andsubcutaneous, as well as inhalation, suppository, or transdermal. Thevaccine may be administered by any means that includes, but is notlimited to, syringes, nebulizers, misters, needleless injection devices,or microprojectile bombardment gene guns.

Alternative routes of application that are feasible are by topicalapplication as a drop, spray, gel or ointment to the mucosal epitheliumof the eye, nose, mouth, anus, or vagina, or onto the epidermis of theouter skin at any part of the body; by spray as aerosol or powder.Alternatively, application may be via the alimentary route, by combiningwith the food, feed or drinking water, e.g., as a powder, a liquid, ortablet, or by administration directly into the mouth as a liquid, a gel,a tablet, or a capsule, or to the anus as a suppository. The preferredapplication route is by intramuscular or by subcutaneous injection.

The vaccine according to the invention may be in several forms, e.g., aliquid, a gel, an ointment, a powder, a tablet, or a capsule, dependingon the desired method of application to the target. The scheme of theapplication of the vaccine according to the invention to the targetmammal may be in single or multiple doses, which may be given at thesame time or sequentially, in a manner compatible with the dosage andformulation, and in such an amount as will be immunologically effective.

Challenge Model

In order to effectively study and evaluate the pathogenic mechanisms ofthe bacteria and the defense mechanisms of the host mammals, and therebyto advance the vaccine art and improve vaccine products, an effectivechallenge model should be employed.

A challenge model, for example, may be based upon the percentage of testanimals that demonstrate persistent and severe clinical symptomscommonly associated with the disease.

Several other cellular diagnostic methods exist to determine thepresence of infection. For example, the presence of infection may bedetermined by direct or indirect immunofluorescence. Other methods todetect infection include staining, e.g., Giemsa, Wright/Giemsa. AcridineOrange can also be utilized to stain the organisms.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are hereby wholly incorporated by reference.

For a clearer understanding of the invention, examples are set forthbelow. These examples are merely illustrative and are understood to notlimit the scope or underlying principles of the invention in any way.Indeed, various modifications of the invention, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the examples set forth hereinbelow and the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

EXAMPLES Example 1 Identification and Characterization of Neorickettsiaristicii Isolates from Oregon, New York and Michigan

A blood sample was obtained from a horse in Oregon showing clinicalsigns of Potomac Horse Fever. We found the Oregon (OR) sample to bepositive for N. risticii by PCR and by immunofluorescent antibodytesting (IFA). We discovered this unique N. risticii Oregon strain to bedistinct from the 90-12 strain. When tested by IFA, it did not reactwith a monoclonal antibody to the 90-12 strain.

Horses vaccinated with the 90-12 strain of N. risticii (studyE-05-09-PHF) were challenged with the N. risticii Oregon. The vaccinatedhorses were not protected from disease. Serologically, the 90-12vaccinated horses had no detectable antibody response to N. risticiiOregon after vaccination, and most of the unvaccinated control horseschallenged with N. risticii Oregon did not have a detectable antibodyresponse to the 90-12 strain 23 days after challenge.

A blood sample was obtained from a horse in New York showing clinicalsigns of Potomac Horse Fever. Unlike the corresponding Oregon isolatedisclosed above, the New York (NY) sample was shown to react with themonoclonal antibody to the 90-12 strain by IFA. When horses vaccinatedwith the 90-12 strain of N. risticii (study E-01-09-PHF) were challengedwith N. risticii New York, the vaccinated horses showed a significantreduction in clinical disease and bacterial isolation.

A blood sample was obtained from a horse in Michigan showing clinicalsigns of Potomac Horse Fever. We found the Michigan (MI) sample to bepositive for N. risticii by PCR and by immunoflorescent antibodytesting. We discovered this unique N. risticii strain to be distinctfrom the 90-12 strain. When tested by Western Blot, it did not reactwith a monoclonal antibody to the 90-12 strain.

Example 2 N. risticii Host Animal Protection Studies

TABLE 1 PHF Vaccine Formulations Component Lot Number Volume E-13-06-PHFVaccine N. risticii (90-12) antigen 120306 300 mL Havlogen Adjuvant 024200 mL PBS #2 095 1276 mL Glycerin 005 200 mL EDTA (240 mM) 006 20 mLThimerosal (10% stock solution) 025 2 mL Phenol Red (1% stock solution)005 2 mL Total Volume: 2000 mL E-01-09-PHF Vaccine N. risticii (90-12)antigen 101608 52 mL Carbopol 971P Adjuvant 002 300 mL PBS #2 436 2642mL Thimerosal (10% stock solution) 043 2.95 mL Phenol Red (1% stocksolution) 008 3 mL Total Volume: 3000 mL E-05-09-PHF Vaccine N. risticii(90-12) antigen 013009 48 mL Carbopol 971P Adjuvant 003 200 mL PBS #2504 1748 mL Thimerosal (10% stock solution) 043 1.95 mL Phenol Red (1%stock solution) 008 1.95 mL Total Volume: 2000 mL E-03-10-PHF Vaccine N.risticii (Oregon) antigen 120809 5.2 mL Carbopol 971P Adjuvant 003 10 mLPBS #2 601 84.6 mL Thimerosal (10% stock solution) 062 0.095 mL PhenolRed (1% stock solution) 008 0.095 mL Total Volume: 100 mL

Example 2a 90-12 Vaccination/90-12 Challenge (Study E-13-06-PHF)

The purpose of this study was to evaluate the efficacy of an inactivatedvaccine containing the 90-12 strain of N. risticii, against challengewith the homologous 90-12 N. risticii isolate, in host animals.

Eight month-old horses, antibody negative to N. risticii, wererandomized into two treatment groups, with fifteen horses per group. Thehorses were vaccinated twice intramuscularly three weeks apart with 1.0mL of vaccine containing the inactivated 90-12 strain of N. risticii(Lot 120306), or with 1.0 mL of a placebo vaccine. The horses werechallenged with the 90-12 strain of N. risticii at twenty-one daysfollowing the second vaccination. Horses were observed for 24 dayspost-challenge for clinical signs of PHF, and whole blood samples werecollected daily from 6 to 24 days post-challenge for bacterial isolationand white blood cell (WBC) counts. The clinical signs of PHF diseaseinclude rectal body temperature of >1.5° F. over baseline temperature(established as the average of the rectal temperatures for 3 days priorto challenge), anorexia, depression, or diarrhea, and laminitis.

Clinical Disease

Horses that exhibited at least one clinical sign of PHF post-challengewere classified as affected. Fifteen of the fifteen (100%)placebo-vaccinated control horses were affected. None of the fifteen(0%) horses vaccinated with N. risticii 90-12 vaccine were affected.

The prevented fraction was 1.00 with a 95% confidence interval of [0.77,1.00], therefore the data supports the claim of “aid in the preventionof disease caused by N. risticii”.

N. risticii Bacteremia

Horses with a positive isolation from a buffy coat sample wereclassified as affected. Fifteen of the fifteen (100%) placebo-vaccinatedcontrol horses were bacteremic for 5-8 days following challenge. Six ofthe fifteen (40%) horses vaccinated with N. risticii 90-12 vaccine werebacteremic for only one day following challenge.

The prevented fraction was 0.60 with a 95% confidence interval of [0.32,0.80], therefore the data supports the claim of “aid in the preventionof bacteremia caused by N. risticii”.

Conclusion

The data demonstrated that inactivated N. risticii 90-12 strain vaccineis protective against challenge with homologous N. risticii strain90-12.

Example 2b 90-12 Vaccination/NY Challenge (Study E-01-09-PHF)

The purpose of this study was to evaluate the efficacy of an inactivatedvaccine containing the 90-12 strain of N. risticii, against challengewith the heterologous NY N. risticii strain, in host animals.

Eleven month-old horses, antibody negative to N. risticii, wererandomized into two treatment groups, with fifteen horses per group. Thehorses were vaccinated twice intramuscularly three weeks apart with 1.0mL of vaccine containing the inactivated 90-12 strain of N. risticii(Lot 101608), or with 1.0 mL of a placebo vaccine. The horses werechallenged with the NY strain of N. risticii fourteen days following thesecond vaccination. Horses were observed for 21 days following challengefor clinical signs of disease associated with PHF, and whole bloodsamples were collected daily from 6 to 18 days post-challenge forbacterial isolation. The clinical signs of PHF disease include rectalbody temperature of >1.5° F. over baseline temperature (established asthe average of the rectal temperatures for 3 days prior to challenge),anorexia, depression, diarrhea, colic or laminitis, when associated withthe other clinical signs of PHF.

Clinical Disease

Horses that exhibited at least one clinical sign of PHF post-challengeon at least one day post-challenge were classified as affected.

The severity of clinical disease was considered to be the number ofpost-challenge days with at least one clinical sign, and was analyzedusing the Wilcoxon Rank Sum test. The mitigated fraction was 0.54 with alower confidence interval of 0.17, therefore the data supports the claimof “aid in the reduction of severity of clinical disease caused by N.risticii”.

N. risticii Bacteremia

Horses with a positive isolation from a buffy coat sample wereclassified as affected. The placebo-vaccinated control horses had anaverage of 6.4 days of positive isolations following challenge. Thehorses vaccinated with inactivated N. risticii 90-12 strain vaccine hadan average of 3.5 days of positive isolations following challenge.

The duration of bacteremia was analyzed using the Wilcoxon Rank Sumtest. The mitigated fraction was 0.59 with a lower confidence intervalof 0.24, therefore the data supports the claim of “aid in the reductionof bacteremia caused by N. risticii”.

Conclusion

The data demonstrated that inactivated N. risticii 90-12 strain vaccinereduces the severity of clinical disease and bacteremia, followingchallenge with heterologous N. risticii strain NY.

Example 2c 90-12 Vaccination/OR Challenge (Study E-05-09-PHF)

The purpose of this study was to evaluate the efficacy of an inactivatedvaccine containing only the OR strain of N. risticii, against challengewith the OR N. risticii isolate, in host animals.

Five to six month-old horses, antibody negative to N. risticii, wererandomized into two treatment groups, with fifteen horses per group. Thehorses were vaccinated twice intramuscularly three weeks apart with 1.0mL of the inactivated vaccine containing the inactivated 90-12 strain ofN. risticii (Lot 013009), or with 1.0 mL of a placebo vaccine. Thehorses were challenged with the heterologous OR strain of N. risticiifourteen days following the second vaccination. Horses were observed for21 days post-challenge for clinical signs of PHF, and whole bloodsamples were collected daily from 6 to 18 days post-challenge forbacterial isolation. The clinical signs of PHF disease include rectalbody temperature of >1.5° F. over baseline temperature (established asthe average of the rectal temperatures for 3 days prior to challenge),anorexia, depression, or diarrhea, and additionally colic or laminitis,when associated with the other clinical signs of PHF.

Clinical Disease

Horses that exhibited at least one clinical sign of PHF post-challengewere classified as affected. Twelve of the fifteen (80%)placebo-vaccinated control horses were affected, and twelve of thefifteen (80%) horses vaccinated with inactivated N. risticii 90-12strain vaccine were affected. The inactivated N. risticii 90-12 strainvaccine provided no protection from clinical disease caused byheterologous OR strain N. risticii.

N. risticii Bacteremia

Horses with a positive isolation from a buffy coat sample wereclassified as affected. Two of the fifteen (13%) placebo-vaccinatedcontrol horses had a positive isolation on one day following challenge.Eight of the fifteen (53%) horses vaccinated with inactivated N.risticii 90-12 strain vaccine had a positive isolation on at least oneday following challenge. The inactivated N. risticii 90-12 strainvaccine provided no protection from bacteremia caused by heterologous ORstrain N. risticii.

Conclusion

The data demonstrates that the inactivated N. risticii 90-12 strainvaccine did not protect against, or reduce the severity of, clinicaldisease or bacteremia following challenge with heterologous N. risticiistrain OR.

Example 2d OR Vaccination 10R Challenge (Study E-03-10-PHF)

The purpose of this study was to evaluate the efficacy of an inactivatedvaccine containing only the OR strain of N. risticii, against challengewith the homologous OR N. risticii isolate, in host animals.

Ten to twelve month-old horses, antibody negative to N. risticii, wereenrolled in the study and were randomized into treatment groups, witheight horses per group. The horses were vaccinated twice intramuscularlythree weeks apart with 1.0 mL of the inactivated vaccine containing theOR strain of N. risticii (Lot 120809), or with 1.0 mL of a placebovaccine. The horses were challenged with the OR strain of N. risticii attwenty days following the second vaccination. Horses were observed for24 days post-challenge for clinical signs of PHF, until resolution ofthe clinical signs, and whole blood samples were collected daily from 7to 18 days post-challenge for bacterial isolation. Horses were weighedprior to challenge and following the post-challenge period, as anadditional non-subjective indication of illness, anorexia, and diarrheacaused by PHF, and percent weight gains or losses during thepost-challenge period were determined. The clinical signs of PHF diseaseinclude rectal body temperature of >1.5° F. over baseline temperature(established as the average of the rectal temperatures for 4 days priorto challenge), anorexia, depression, or diarrhea, and additionally colicor laminitis, when associated with the other clinical signs of PHF.

Clinical Disease

Horses that exhibited at least one clinical sign of PHF post-challengewere classified as affected. Seven of the eight (88%) placebo-vaccinatedcontrol horses were affected. Three of the eight (38%) horses vaccinatedwith experimental N. risticii OR strain vaccine were affected.

The Fisher test p-value was 0.0594. The proportion of affected horsesvaccinated with N. risticii OR strain vaccine was marginally less thanthe proportion of horses vaccinated with placebo.

Severity of Disease

To evaluate disease severity, the number of post-challenge days with atleast one clinical sign of PHF was analyzed using the Wilcoxon Rank Sumtest. The p-value was 0.0485, therefore the number of post-challengedays with at least one clinical sign of PHF for horses vaccinated withN. risticii OR strain vaccine was marginally less than for theplacebo-vaccinated horses.

The Wilcoxon Rank Sum test was also used to evaluate the number ofclinical signs of PHF present during the post-challenge period, as anadditional measure of disease severity. The p-value was 0.0485,indicating that the total number of clinical signs of PHF present duringthe post-challenge period was marginally less for the horses vaccinatedwith experimental N. risticii OR strain vaccine than for theplacebo-vaccinated control horses.

N. risticii Bacteremia

Horses with a positive isolation from a buffy coat sample wereclassified as affected. Two of the eight (25%) placebo-vaccinatedcontrol horses had a positive isolation on one day following challenge.One of the eight (13%) horses vaccinated with experimental N. risticiiOR strain vaccine had a positive isolation on one day followingchallenge.

The Fisher test p-value was 0.3354, therefore there was no statisticaldifference in bacteremia between the horses vaccinated with experimentalN. risticii OR strain vaccine and the placebo-vaccinated horses.

Weight

Horses were weighed prior to and following challenge. Four of the eightplacebo-vaccinated control horses lost weight during the post-challengeperiod, and the average percent of body weight gain for all eight horseswas only 0.3%. The horses vaccinated with experimental N. risticii ORstrain vaccine had an average percent weight gain of 5.2%, and none ofthe eight vaccinated horses lost weight.

Conclusion

This data demonstrates that vaccination of horses with N. risticii ORstrain vaccine reduces the incidence and severity of clinical PHFdisease, compared to placebo-vaccinated control horses, followingchallenge with homologous OR strain N. risticii. Table 2 provides acomparison of the clinical results of sixteen horses challenged with N.risticii Oregon, eight of which received a vaccine containing the N.risticii Oregon isolate and eight of which received a placebo.

TABLE 2 Control Vaccinated Horses Horses Treatment Group (Placebo)(Oregon strain) No. of Horses with ≧1 Clinical Sign of PHF 7/8 3/8 No.of Horses with Fever 5/8 1/8 No. of Horses with Anorexia 4/8 2/8 No. ofHorses with Depression 1/8 0/8 No. of Horses with Diarrhea 3/8 1/8 No.of Horses with Laminitis 1/8 0/8 No. of Horses with Bacteremia 2/8 1/8Average % Weight Gain 0.3 5.2These data show that vaccination of horses with N. risticii Oregonstrain vaccine reduces the incidence and severity of clinical PHF,compared to placebo-vaccinated control horses.

Example 3 Serological Cross-Reactivity of N. risticii Strains

Immunofluorescent antibody (IFA) testing was performed on sera collectedfrom horses following vaccination and/or challenge with various strainsof N. risticii, to measure the antibody reactivity against the fivedifferent strains of N. risticii. For the IFA assay, fixed felineembryonic fibroblast (FEF) cells, infected with one of the N. risticiistrains (OR, 90-12, NY, MI, or 25-D), were reacted with serial dilutionsof the equine serum samples. The plates were read using a fluorescencemicroscope for positive fluorescence, as indicated by bright green,specific cytoplasmic staining. The antibody titer was the reciprocal ofthe highest serum dilution that showed positive fluorescence.

The IFA results show that for most strains of N. risticii there is someserological cross-reactivity to other strains of N. risticii, but thereappears to be very little cross-reactivity in sera from horseschallenged with the OR strain of N. risticii, to other strains of N.risticii. The OR strain of N. risticii appears to be highly uniqueantigenically.

TABLE 3 Oregon Challenged Horses (21, 22 or 24 days post-challenge)Oregon New York 90-12 Michigan Study Horse IFA IFA IFA IFA 25D IFA OSU*IFA No. ID Results Results Results Results Results Results E-05-09 1211280 80 40 40 80 40 E-05-09 123 2560 320 160 320 320 2560 E-05-09 126640 80 <20 80 80 320 E-05-09 127 320 40 <20 40 40 40 E-05-09 128 1280 40<20 <20 20 <20 E-05-09 130 640 <20 <20 <20 40 <20 E-05-09 132 640 80 <2020 40 <20 E-05-09 133 320 <20 <20 <20 <20 <20 E-05-09 136 1280 <20 <20<20 <20 <20 E-05-09 137 1280 40 <20 40 <20 <20 E-05-09 138 640 20 <20 4040 <20 E-05-09 140 320 <20 <20 <20 <20 <20 E-05-09 144 640 20 <20 <20<20 <20 E-05-09 147 2560 80 <20 80 160 <20 E-05-09 148 640 40 <20 160 40<20 E-07-08 2 1280 <20 <20 <20 <20 NT E-07-08 5 1280 <20 <20 <20 <20 NTE-07-08 10 640 <20 <20 <20 <20 NT E-07-08 22 640 <20 <20 <20 <20 NTE-07-08 11 1280 80 <20 40 <20 NT E-07-08 12 2560 80 <20 <20 160 NTE-07-08 17 640 <20 <20 <20 <20 NT E-07-08 21 640 <20 <20 <20 <20 NTE-10-10 328 80 <20 <20 <20 <20 NT E-10-10 331 640 <20 <20 <20 <20 NTE-10-10 333 160 40 <20 <20 40 NT E-10-10 337 320 80 <20 <20 <20 NTE-10-10 336 1280 160 <20 40 <20 NT E-10-10 338 80 <20 <20 <20 <20 NTE-10-10 339 640 80 <20 40 80 NT E-03-10 162 640 80 80 40 80 NT E-03-10165 640 <20 20 <20 80 NT E-03-10 167 1280 80 40 40 20 NT E-03-10 173 64080 80 80 <20 NT E-03-10 177 320 40 40 40 <20 NT E-03-10 179 640 40 40 4040 NT E-03-10 214 640 80 80 80 80 NT E-03-10 216 640 40 40 40 40 NTAverage: 859 53 27 42 46 208

TABLE 4 Oregon Vaccinated and Challenged Horses (24 days post-challenge)Oregon New York 90-12 Michigan Study Horse IFA IFA IFA IFA 25D IFA No.ID Results Results Results Results Results E-03-10 171 1280 640 320 640640 E-03-10 175 2560 640 640 1280 1280 E-03-10 204 1280 320 160 320 640E-03-10 208 1280 320 320 320 640 E-03-10 213 1280 320 320 320 320E-03-10 217 1280 320 320 320 640 E-03-10 218 1280 320 80 80 640 E-03-10219 2560 2560 2560 2560 2560 Average: 1600 680 590 730 920

TABLE 5 New York Challenged Horses (22 days post-challenge) Oregon NewYork 90-12 Michigan Study Horse IFA IFA IFA IFA 25D IFA OSU IFA No. IDResults Results Results Results Results Results E-07-08 4 160 640 320320 640 NT E-07-08 6 40 160 80 <20 <20 NT E-07-08 14 160 1280 320 640640 NT E-07-08 19 40 320 160 160 320 NT E-07-08 9 80 320 160 160 320 NTE-07-08 15 80 640 160 320 640 NT E-07-08 23 160 1280 320 320 320 NTE-07-08 24 80 1280 640 320 320 NT E-01-09 74 <20 1280 1280 320 640 640E-01-09 76 160 1280 1280 640 640 640 E-01-09 77 80 640 640 320 320 160E-01-09 78 80 640 1280 320 640 320 E-01-09 79 <20 640 1280 640 640 320E-01-09 81 160 320 640 160 320 80 E-01-09 87 160 640 1280 640 640 320E-01-09 92 <20 1280 1280 1280 640 1280 E-01-09 93 <20 2560 640 640 1280640 E-01-09 96 640 1280 640 1280 1280 1280 E-01-09 97 <20 1280 640 320640 320 E-01-09 98 160 2560 2560 1280 2560 1280 E-01-09 100 <20 2560 640320 640 320 E-01-09 102 80 1280 1280 640 320 320 E-01-09 106 <20 2560640 <20 <20 320 Average: 105 1162 790 481 627 549

TABLE 6 90-12 Challenged Horses (21 days post-challenge) Oregon New York90-12 Michigan Study Horse IFA IFA IFA IFA 25D IFA No. ID ResultsResults Results Results Results E-08-08 27 80 1280 640 320 320 E-08-0830 640 1280 2560 1280 320 E-08-08 32 80 1280 1280 640 640 E-08-08 40 6402560 5120 2560 5120 E-08-08 45 320 2560 2560 2560 2560 E-08-08 46 6405120 2560 2560 2560 E-08-08 48 320 2560 2560 1280 2560 E-08-08 49 1601280 640 640 1280 E-08-08 52 160 1280 1280 640 1280 E-08-08 53 160 1280640 640 1280 E-08-08 55 80 1280 640 640 320 E-08-08 57 80 1280 640 640640 E-08-08 59 160 2560 5120 2560 2560 E-08-08 66 640 2560 2560 51205120 E-08-08 71 40 640 640 640 640 Average: 280 1920 1963 1515 1813

TABLE 7 90-12 Vaccinated and Challenged Horses (21 days post-challenge)Oregon New York 90-12 Michigan Study Horse IFA IFA IFA IFA 25D IFA No.ID Results Results Results Results Results E-08-08 26 1280 10240 102405120 2560 E-08-08 29 1280 10240 10240 2560 5120 E-08-08 34 1280 51205120 2560 5120 E-08-08 35 640 10240 10240 2560 5120 E-08-08 36 128010240 10240 5120 10240 E-08-08 37 1280 10240 10240 10240 10240 E-08-0838 1280 10240 10240 5120 5120 E-08-08 39 2560 10240 10240 10240 10240E-08-08 41 1280 5120 2560 5120 10240 E-08-08 44 2560 10240 10240 1024010240 E-08-08 50 1280 10240 10240 5120 10240 E-08-08 51 640 5120 51205120 10240 E-08-08 60 1280 2560 5120 5120 2560 E-08-08 64 1280 512010240 10240 5120 E-08-08 69 2560 5120 10240 10240 10240 Average: 14518021 8704 6315 7509

TABLE 8 Michigan Challenged Horses (21 days post-challenge) Oregon NewYork 90-12 Michigan Study Horse IFA IFA IFA IFA 25D IFA No. ID ResultsResults Results Results Results E-10-10 326 320 640 320 640 1280 E-10-10327 <20 <20 <20 80 80 E-10-10 335 320 2560 640 640 1280 Average: 2181072 325 453 880

TABLE 9 25D Challenged Horses (21 days post-challenge) Oregon New York90-12 Michigan Study Horse IFA IFA IFA IFA 25D IFA No. ID ResultsResults Results Results Results E-10-10 329 40 160 40 40 320 E-10-10 332<20 160 80 40 320 E-10-10 334 <20 320 160 160 640 E-10-10 340 <20 160 4080 640 Average: 21 200 80 80 480 NT = Not Tested, NA = Not Applicable<20 assigned a value of 15 for average titer calculations Antigen Lots:Oregon Lot 012010, 90-12 Lot 101910, Michigan Lot 100510, New York Lot101310, 25-D Lot 101210 *OSU = Oregon State University VeterinaryDiagnostic Laboratory

Example 4 Western Blot Analysis of N. risticii Strains

Western Blots were performed to evaluate the antibody reactivity andantigen banding patterns of rabbit and horse serum against therecombinant 85 kDa protein of the N. risticii 90-12 strain with FEFcells used to propagate N. risticii and against the 90-12, OR, NY, MIand 25-D strains of N. risticii. Rabbit serum lot 02x1808a was a pool ofsera collected from four rabbits following hyperimmunization withpurified recombinant 85 kDa protein from the 90-12 strain of N.risticii. Horse serum lot 112310 was a pool of sera collected from fourhorses following challenge with the 90-12, OR, NY, or MI strains of N.risticii. Horse serum lot 012809 was collected from a horse that had notpreviously been exposed to, vaccinated or challenged with N. risticii,and served as a negative control to identify non-specific reactivity toFEF cells and N. risticii.

The pooled serum from rabbits immunized with the recombinant 85 kDaprotein of the N. risticii 90-12 strain reacted with the 85 kDarecombinant protein on the Western Blot at the expected molecularweight, and at the same approximate molecular weight for the 90-12strain of N. risticii. (FIG. 1). The serum reacted at differing, lowermolecular weight bands of approximately 50, 55, 60 and 55 kDa for theOR, NY, MI and 25-D strains of N. risticii, respectively. No reactionswere observed for the FEF cells.

The pooled serum from horses challenged with one of four strains of N.risticii, reacted with the recombinant 85 kDa protein at a lower thanexpected molecular weight of approximately 33 kDa on the Western Blot.(FIG. 2). The predominant unique bands for strain 90-12 of N. risticiihad molecular weights of approximately 177, 89, and 22 kDa. The serumreacted at differing molecular weight bands of approximately 47 for theOR strain, 180 and 39 kDa for the NY strain, 180 and 31 kDa for the MIstrain, and 79 kDa for the 25-D strain of N. risticii. No reactions wereobserved for the FEF cells. These banding patterns indicate that thechallenge strains are distinctive and unique.

The bands that appear at the molecular weights of approximately 61-63kDa and 54-56 kDa of the N. risticii strains are non-specific bands thatalso react with seronegative horse serum.

Example 5 DNA Sequencing Analysis of N. risticii Strains

GeneWiz generated consensus sequences for MAH for the strain specificantigen (SSA) locus of the 90-12, OR, NY, MI and 25-D strains of N.risticii. The sequences were analyzed and compared with each other andwith the currently published sequences for the N. risticii Illinoisstrain. Both nucleotide sequences and predicted protein sequences wereanalyzed using the National Center for Biotechnology Information (NCB')Basic Local Alignment Search Tool (BLAST). In addition, DNASTAR™LASERGENE MEGALIGN analysis software (version 8.0.2) was used to alignand compare sequences with one another.

TABLE 10 DNA Size comparison: N. risticii Strain SSA Locus SSA-1 SSA-2SSA-3 Illinois ~6600 bp 1560 bp 1539 bp 1302 bp (acc# CP001431) 90-12  6356 bp 1887 bp 1539 bp 1614 bp OR   6871 bp 1422 bp 1464 bp 1305 bp(inverted) NY   6916 bp 1638 bp 1539 bp 1614 bp MI   6917 bp 1869 bp1410 bp 1458 bp 25-D   6620 bp 1620 bp 1539 bp 1302 bp

The BLAST results differ slightly from those obtained using the DNASTAR™LASERGENE MEGALIGN program, which indicates that the NY strain sharesthe highest identity with the published Illinois strain. MEGALIGNsoftware uses the CLUSTALW, SLOW_ACCURATE algorithm for aligningmultiple sequences (FIGS. 4-7), while the NCBI BLAST search uses theBLASTN and BLASTP algorithms (TABLE 11). In FIG. 7 the nucleic acidsequence for OR SSA#3 is the reversed complimentary sequence from thewild type sequence in order to match it with the genes of the comparedN. risticii strains.

TABLE 11 BLAST Analysis Results Summary: Top Blast Hit Genebank StrainPortion analyzed (% identity) Accession No. 90-12 whole fragment (DNA)92.5% to Illinois CP00143 (94% to 90-12: 85 kd)* SSA1 (amino acid 82.5%to 90-12: 85 kd AAC31428 sequence) SSA2 (amino acid 99.0% to IllinoisYP_0030 sequence) (37% to 90-12: 85 kd) SSA3 (amino acid 94.2% toIllinois YP_0030 sequence) OR whole fragment (DNA) 87.3% to IllinoisCP00143A SSA1 (amino acid 52.6% to Illinois YP_0030 sequence) SSA2(amino acid 63.5% to Illinois YP_0030 sequence) SSA3 (amino acid 88.1%to Illinois YP_0030 sequence) NY whole fragment (DNA) 94.0% to IllinoisCP00143 SSA1 (amino acid 86.3% to Illinois YP_0030 sequence SSA2 (aminoacid 98.8% to Illinois YP_0030 sequence) SSA3 (amino acid 94.2% toIllinois YP_0030 sequence) MI whole fragment (DNA) 97.0% to IllinoisCP00143 SSA1 (amino acid 69.0% to 25-D: 50 kd AAC31427 sequence) (67% toIllinois) SSA2 (amino acid 90.9% to Illinois YP_0030 sequence) SSA3(amino acid 85.6% to Illinois YP_0030 sequence) 25-D whole fragment(DNA) 96.3% to Illinois CP00143A SSA1 (amino acid 98.0% to 25-D: 50 kdAAC31427 sequence) SSA2 (amino acid 99.8% to Illinois YP_0030 sequence)ssa3 (amino acid seq.) 99.8% to Illinois YP_0030 *Accession AF059673(strain 90-12) 85 kd SSA gene nucleotide sequence is smaller than wholefragment sequenced.

Biological Deposit

Cultures of the following biological materials have been deposited withthe following international depository: American Type Culture Collection(ATCC) 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A.,under conditions that satisfy the requirements of the Budapest Treaty.

Organism Accession No. Date of Deposit Neorickettsia risticii PTA-11232Jul. 27, 2010 Oregon Neorickettsia risticii PTA-11231 Jul. 27, 2010 NewYork Neorickettsia risticii PTA-11404 Oct. 13, 2010 Michigan

SEQUENCE LISTING TABLE SEQ ID NO: STRAIN SS# Type  1 Illinois 1 AA  2Oregon 1 AA  3 25-D 1 AA  4 90-12 1 AA  5 Michigan 1 AA  6 New York 1 AA 7 Illinois 2 AA  8 New York 2 AA  9 90-12 2 AA 10 25-D 2 AA 11 Oregon 2AA 12 Michigan 2 AA 13 Illinois 3 AA 14 New York 3 AA 15 90-12 3 AA 1625-D 3 AA 17 Michigan 3 AA 18 Oregon 3 AA 19 Illinois 3 NA 20 New York 3NA 21 90-12 3 NA 22 25-D 3 NA 23 Michigan 3 NA 24 Oregon 3 NA 25 Oregon1 NA 26 Oregon 2 NA 27 New York 1 NA 28 New York 2 NA 29 Michigan 1 NA30 Michigan 2 NA 31 Illinois 1 NA 32 Illinois 2 NA 33 25-D 1 NA 34 25-D2 NA 35 90-12 1 NA 36 90-12 2 NA AA is an amino acid sequence; NA is anucleic acid sequence.

It is to be understood that all base sizes or amino acid sizes, and allmolecular weight or molecular mass values, provided to describe nucleicacids and polypeptides according to the invention are approximate withinconventional measurement variations.

We claim:
 1. An isolated Neorickettsia risticii (N. risticii) bacteriumthat is the N. risticii Oregon isolate having the ATCC deposit numberPTA-11232.
 2. A composition comprising an immunogenically effectiveamount of a N. risticii bacterium according to claim 1 and a carrier. 3.The composition of claim 2, further comprising a different N. risticiiisolate.
 4. A vaccine comprising an immunogenically effective amount ofa N. risticii bacterium according to claim 1 and a carrier; wherein saidvaccine reduces the incidence and severity of clinical Potomac HorseFever disease in a horse when that horse is challenged with an N.risticii Oregon strain.